Mucus obstruction is a key pathogenic step in many major human airways diseases, including COPD and CF. As part of the drug discovery process, it is extremely valuable to have a small animal model that will accelerate the transition from in vitro studies of target identification/validation to early proof-of-concept studies in humans. The ?ENaC mouse has been engineered to exhibit many of the features of muco- obstructive human airways diseases, expressing a disease-initiating transgene (?ENaC) that drives airway surface dehydration. The airways dehydration of the ?ENaC mouse triggers a sequence of intraluminal airways mucus plaque/plug formation, airways inflammation with macrophage activation, airways remodeling, bacterial infection, and emphysema. In Project II, we propose to contribute to the tPPG goals and needs for drug development as follows: 1) test in vivo the novel biophysical/biochemical formulation of a two-gel mucus clearance system, focusing on relationships between the rate of delivery of hypertonic saline (HS) and mucus clearance responses; 2) develop novel measures of mucus properties that will serve as validated biomarkers to predict the magnitude of the airway mucus burden, i.e., our therapeutic target, utilizing PENaC mouse lines of differing ainways Na+ transport/airway surface dehydration; 3) test the hypothesis that secreted mucins (MUC5AC, MUCSB) are therapeutic targets with favorable risk:benefit ratios by genetic studies of MUC5AC and MUCSB -/- crosses with ?ENaC mice and pharmacologic (novel ketolides/macrolides) approaches; and 4) test the hypothesis that bacterial infection of mucus-obstructed airways can be prevented or reversed by hydration/mucolytic therapies. Thus, Project II should provide important in vivo data as to the validity of our novel two-gel model of mucus transport, identify biomarkers for pharmacodynamic studies of mucus modification to speed drug development in both animals and the clinic, and assist Core A to identify relevant targets and novel therapies of muco-obstructive lung disease.